IL-22 promotes mucin-type O-glycosylation and MATH1+ cell-mediated amelioration of intestinal inflammation.

The interleukin (IL)-22 cytokine can be protective or inflammatory in the intestine. It is unclear if IL-22 receptor (IL-22Ra1)-mediated protection involves a specific type of intestinal epithelial cell (IEC). By using a range of IEC type-specific Il22Ra1 conditional knockout mice and a dextran sulfate sodium (DSS) colitis model, we demonstrate that IL-22Ra1 signaling in MATH1+ cells (goblet and progenitor cells) is essential for maintaining the mucosal barrier and intestinal tissue regeneration. The IL-22Ra1 signaling in IECs promotes mucin core-2 O-glycan extension and induces beta-1,3-galactosyltransferase 5 (B3GALT5) expression in the colon. Adenovirus-mediated expression of B3galt5 is sufficient to rescue Il22Ra1IEC mice from DSS colitis. Additionally, we observe a reduction in the expression of B3GALT5 and the Tn antigen, which indicates defective mucin O-glycan, in the colon tissue of patients with ulcerative colitis. Lastly, IL-22Ra1 signaling in MATH1+ progenitor cells promotes organoid regeneration after DSS injury. Our findings suggest that IL-22-dependent protective responses involve O-glycan modification, proliferation, and differentiation in MATH1+ progenitor cells.

Aryl Hydrocarbon Receptor Activation Coordinates Mouse Small Intestinal Epithelial Cell Programming.

In the face of mechanical, chemical, microbial, and immunologic pressure, intestinal homeostasis is maintained through balanced cellular turnover, proliferation, differentiation, and self-renewal. Here, we present evidence supporting the role of the aryl hydrocarbon receptor (AHR) in the adaptive reprogramming of small intestinal gene expression, leading to altered proliferation, lineage commitment, and remodeling of the cellular repertoire that comprises the intestinal epithelium to promote intestinal resilience. Ahr gene/protein expression and transcriptional activity exhibit marked proximalHI to distalLO and cryptHI to villiLO gradients. Genetic ablation of Ahr impairs commitment/differentiation of the secretory Paneth and goblet cell lineages and associated mucin production, restricts expression of secretory/enterocyte differentiation markers, and increases crypt-associated proliferation and villi-associated enterocyte luminal exfoliation. Ahr-/- mice display a decrease in intestinal barrier function. Ahr+/+ mice that maintain a diet devoid of AHR ligands intestinally phenocopy Ahr-/- mice. In contrast, Ahr+/+ mice exposed to AHR ligands reverse these phenotypes. Ligand-induced AHR transcriptional activity positively correlates with gene expression (Math1, Klf4, Tff3) associated with differentiation of the goblet cell secretory lineage. Math1 was identified as a direct target gene of AHR, a transcription factor critical to the development of goblet cells. These data suggest that dietary cues, relayed through the transcriptional activity of AHR, can reshape the cellular repertoire of the gastrointestinal tract.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

Transcriptional Regulation of Math1 by Aryl Hydrocarbon Receptor: Effect on Math1+ Progenitor Cells in Mouse Small Intestine.

The physiological roles of aryl hydrocarbon receptor (AhR) in the small intestine have been revealed as immunomodulatory and barrier functions. However, its contributions to cell fate regulation are incompletely understood. The Notch-activated signaling cascade is a central component of intestinal cell fate determinations. The lateral inhibitory mechanism governed by Notch directs cell fates toward distinct cell lineages (i.e., absorptive and secretory cell lineages) through its downstream effector, mouse atonal homolog 1 (MATH1). An investigation employing cell lines and intestinal crypt cells revealed that AhR regulates Math1 expression in a xenobiotic response element (XRE)-dependent manner. The AhR-Math1 axis was further addressed using intestinal organoids, where AhR-Math1 and HES1-Math1 axes appeared to coexist within the underlying Math1 transcriptional machinery. When the HES1-Math1 axis was pharmacologically suppressed, ß-naphthoflavone-mediated AhR activation increased the number of goblet and Math1+ progenitor cells in the organoids. The same pharmacological dissection of the AhR-Math1 axis was applied in vivo, demonstrating an enhanced number of Math1+ progenitor cells in the small intestine following AhR activation. We report here that AhR-Math1 is a direct transcriptional axis with effects on Math1+ progenitor cells in the small intestine, highlighting a novel molecular basis for fine-tuning Notch-mediated cell fate regulation.

Retinoic acid induces differentiation of cochlear neural progenitor cells into hair cells / O ácido retinoico induz a diferenciação das células progenitoras neurais cocleares em células ciliadas

Abstract Introduction: Inner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells. Objective: To investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation. Methods: Cochlear neural progenitor cells were cultured and induced in DMEM/F12 + RA (10−6M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu. Results: The nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p <0.05). The ratios of Brdu+-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased. Conclusion: Retinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.

Resumo Introdução: As células progenitoras da orelha interna têm potencial para diferenciação multidirecional. O ácido retinoico é uma condição importante para o desenvolvimento da orelha interna. O bloqueio da via de sinalização do ácido retinoico no órgão de Corti pode reduzir significativamente o número de células ciliadas. Portanto, acreditamos que o ácido retinoico pode induzir a regeneração das células ciliadas do ouvido interno. Objetivo: Investigar se as células progenitoras neurais cocleares mantêm as características das células-tronco durante a recuperação e subcultura, se o ácido retinoico pode induzir a transformação de células progenitoras neurais cocleares em células ciliadas in vitro e se o ácido retinoico promove ou inibe a proliferação das células progenitoras durante a diferenciação. Método: As células progenitoras neurais cocleares foram cultivadas e induzidas em DMEM/F12+AR (106M) e, então, foram detectadas as expressões de marcadores das células ciliadas (Math1 e Myosin?a) com o uso de citoquímica por imunofluorescência e real time -polymerase chain reaction e a proliferação de células progenitoras neurais cocleares foi detectada pelo teste Brdu. Resultados: A nestina das células progenitoras neurais cocleares foi expressa positivamente. As proporções de células positivas para Math1 no grupo controle e no grupo experimental foram 1,5% e 63%, respectivamente; as proporções de células positivas para Myosin?a no grupo controle e no grupo experimental foram de 0,96% e 56%, respectivamente (p <0,05). As proporções de células marcadas com Brdu+ no grupo ácido retinoico, grupo PBS e grupo FBS foram de 20,6%, 29,9% e 54,3%, respectivamente; no entanto, a taxa de proliferação no grupo experimental diminuiu. Conclusões: O ácido retinoico pode promover a diferenciação das células progenitoras neurais cocleares em células ciliadas.

Effects of Notch signaling pathway inhibition by dibenzazepine in acute experimental toxoplasmosis.

Notch signaling pathway plays a crucial role in cellular fate across species, being important for the differentiation and development of several cell types. The aim of this study was to evaluate the effect of Notch inhibition pathway by dibenzazepine (DBZ) in histological and inflammatory alterations and, tissue parasitism in acute Toxoplasma gondii infection. For this, C57BL/6 mice were treated with DBZ before infection with T. gondii, and the small intestine, lungs and liver were analyzed. The genes related to Notch signaling pathway were assayed through qPCR in the organs, and cytokine measurement was performed in serum samples. In the small intestine, T. gondii infection impaired the Hes1 and Math1 mRNA expressions, increased the inflammation and decreased goblet and Paneth cell numbers. The DBZ-treatment was able to partially preserve these cells, however, the parasitism and inflammation were not altered. In parallel, the high IL-2, IL-6, TNF and, IFN-γ levels induced by infection were not changed with the DBZ treatment, with the IFN-γ levels even higher. In contrast, in the liver and lungs, the DBZ-treatment diminished parasitism and inflammation. Our results highlight that Notch pathway inhibition in T.gondii infection results in different parasitological and inflammatory outcomes depending on the organ analyzed.

Retinoic acid induces differentiation of cochlear neural progenitor cells into hair cells.

INTRODUCTION: Inner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells. OBJECTIVE: To investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation. METHODS: Cochlear neural progenitor cells were cultured and induced in DMEM/F12+RA (10-6M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu. RESULTS: The nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p<0.05). The ratios of Brdu+-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased. CONCLUSION: Retinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.

Inhibitory Effect of Corilagin on miR-21-Regulated Hepatic Fibrosis Signaling Pathway.

Corilagin is a polyphenol that can be extracted from many medicinal plants and shows multiple pharmacological effects. We aimed to investigate the role of corilagin on miR-21-regulated hepatic fibrosis, especially miR-21-regulated TGF-ß1/Smad signaling pathway, in hepatic stellate LX2 cell line and Sprague-Dawley rats. The mRNA or protein levels of miR-21, Smad7, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), collagen type I alpha 1 (COL1A1), Smad2, Smad3, Smad2/3, p-Smad2, p-Smad3, p-Smad2/3, and transforming growth factor-ß1 (TGF-ß1) in LX2 cells and liver tissues were determined. Furthermore, gain-of and loss-of function of miR-21 in miR-21-regulated TGF-ß1/Smad signaling pathway were analyzed in LX2 cells. Liver tissues and serum were collected for pathological analysis, immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA). Corilagin treatment reduced mRNA or protein levels of miR-21, CTGF, α-SMA, TIMP-1, TGF-ß1, COL1A1, p-Smad2, p-Smad3, and p-Smad2/3 both in vitro and in vivo. While corilagin increased mRNA and protein levels of Smad7 and MMP-9. After gain-of and loss-of function of miR-21, the downstream effectors of miR-21-regulated TGF-ß1/Smad signaling pathway in LX2 cells changed accordingly, and the changes were inhibited by corilagin. Simultaneously, administration of corilagin not only ameliorated pathological manifestation of liver fibrosis but also reduced levels of α-SMA and COL1A1 in liver tissues and TGF-ß1, ALT levels in serum. Corilagin is able to potentially prevent liver fibrosis by blocking the miR-21-regulated TGF-ß1/Smad signaling pathway in LX2 cells and CCl4-induced liver fibrosis rats, which may provide a novel therapeutic strategy for liver fibrosis.

Histological, cellular and behavioural analyses of effects of chemotherapeutic agent cyclophosphamide in the developing cerebellum.

OBJECTIVES: We performed histological, cellular and behavioural analyses of the effects of cyclophosphamide (CTX), a chemotherapeutic drug, in the developing cerebellum and aimed to provide valuable insights into clinical application of CTX in children. MATERIALS AND METHODS: C57BL/6 mice and Math1-dependent GFP expression transgenic mice were used in the research. H&E staining was performed to analyse histological effects of CTX in the cerebellum. Staining for EdU and TUNEL was used to estimate the cell proliferation and apoptosis. Rotarod test and hanging wire test were used to evaluate the behavioural functions. Immunofluorescent staining was used to identify the cell types. The differentiation markers and genes related to Sonic Hedgehog (SHH) signalling were measured via quantitative real-time PCR or immunoblotting. RESULTS: We found that while CTX induced a significant reduction in cell proliferation and increased apoptosis in the EGL in 48 hours, the behavioural functions and the multilayer laminar structure of cerebella were largely restored when the mice grew to adults. Mechanistically, granule neuron progenitors, driven by the SHH signalling, enhanced the capability of proliferation quickly after CTX administration was stopped, which allowed the developing cerebellum to catch up and to gradually replenish the injury. CONCLUSION: The chemotherapeutic agent CTX induces an immediate damage to the developing cerebellum, but the cerebellar multilayer laminar structure and motor function can be largely restored if the agent is stopped shortly after use.

Atoh1 Controls Primary Cilia Formation to Allow for SHH-Triggered Granule Neuron Progenitor Proliferation.

During cerebellar development, granule neuron progenitors (GNPs) proliferate by transducing Sonic Hedgehog (SHH) signaling via the primary cilium. Precise regulation of ciliogenesis, thus, ensures proper GNP pool expansion. Here, we report that Atoh1, a transcription factor required for GNPs formation, controls the presence of primary cilia, maintaining GNPs responsiveness to SHH. Loss of primary cilia abolishes the ability of Atoh1 to keep GNPs in a proliferative state. Mechanistically, Atoh1 promotes ciliogenesis by transcriptionally regulating Cep131, which facilitates centriolar satellite (CS) clustering to the basal body. Importantly, ectopic expression of Cep131 counteracts the effects of Atoh1 loss in GNPs by restoring proper localization of CS and ciliogenesis. This Atoh1-CS-primary cilium-SHH pro-proliferative pathway is also conserved in SHH-type medulloblastoma, a pediatric brain tumor arising from the GNPs. Together, our data reveal how Atoh1 modulates the primary cilium to regulate GNPs development.

Cell proliferation during hair cell regeneration induced by Math1 in vestibular epithelia in vitro.

Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds: supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein (EGFP) was used to track cell division during hair cell transformation. 5-Bromo-2'-deoxyuridine (BrdU) was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.

Jak2-mediated phosphorylation of Atoh1 is critical for medulloblastoma growth.

Treatment for medulloblastoma, the most common malignant brain tumor in children, remains limited to surgical resection, radiation, and traditional chemotherapy; with long-term survival as low as 50-60% for Sonic Hedgehog (Shh)-type medulloblastoma. We have shown that the transcription factor Atonal homologue 1 (Atoh1) is required for Shh-type medulloblastoma development in mice. To determine whether reducing either Atoh1 levels or activity in tumors after their development is beneficial, we studied Atoh1 dosage and modifications in Shh-type medulloblastoma. Heterozygosity of Atoh1 reduced tumor occurrence and prolonged survival. We discovered tyrosine 78 of Atoh1 is phosphorylated by a Jak2-mediated pathway only in tumor-initiating cells and in human SHH-type medulloblastoma. Phosphorylation of tyrosine 78 stabilizes Atoh1, increases Atoh1's transcriptional activity, and is independent of canonical Jak2 signaling. Importantly, inhibition of Jak2 impairs tyrosine 78 phosphorylation and tumor growth in vivo. Taken together, inhibiting Jak2-mediated tyrosine 78 phosphorylation could provide a viable therapy for medulloblastoma.

An Atoh1-S193A Phospho-Mutant Allele Causes Hearing Deficits and Motor Impairment.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix (bHLH) transcription factor that is essential for the genesis, survival, and maturation of a variety of neuronal and non-neuronal cell populations, including those involved in proprioception, interoception, balance, respiration, and hearing. Such diverse functions require fine regulation at the transcriptional and protein levels. Here, we show that serine 193 (S193) is phosphorylated in Atoh1's bHLH domain in vivo Knock-in mice of both sexes bearing a GFP-tagged phospho-dead S193A allele on a null background (Atoh1S193A/lacZ) exhibit mild cerebellar foliation defects, motor impairments, partial pontine nucleus migration defects, cochlear hair cell degeneration, and profound hearing loss. We also found that Atoh1 heterozygous mice of both sexes (Atoh1lacZ/+) have adult-onset deafness. These data indicate that different cell types have different degrees of vulnerability to loss of Atoh1 function and that hypomorphic Atoh1 alleles should be considered in human hearing loss.SIGNIFICANCE STATEMENT The discovery that Atonal homolog 1 (Atoh1) governs the development of the sensory hair cells in the inner ear led to therapeutic efforts to restore these cells in cases of human deafness. Because prior studies of Atoh1-heterozygous mice did not examine or report on hearing loss in mature animals, it has not been clinical practice to sequence ATOH1 in people with deafness. Here, in seeking to understand how phosphorylation of Atoh1 modulates its effects in vivo, we discovered that inner ear hair cells are much more vulnerable to loss of Atoh1 function than other Atoh1-positive cell types and that heterozygous mice actually develop hearing loss late in life. This opens up the possibility that missense mutations in ATOH1 could increase human vulnerability to loss of hair cells because of aging or trauma.

Efficient induction of inner ear hair cell-like cells from mouse ES cells using combination of Math1 transfection and conditioned medium from ST2 stromal cells.

We sought to establish a more efficient technique for induction of inner ear hair cell-like cells (HC-like cells) from embryonic stem cells (ES cells) by using a combination of two previously reported methods; ST2 stromal cell-conditioned medium, known to be favorable for HC-like cell induction (HIST2 method), and ES cells with transfer of the Math1 gene (Math1-ES cells). Math1-ES cells carrying Tet-inducible Math1 were cultured for 14days with doxycycline in conditioned medium from cultures of ST2 stromal cells following formation of 4-day embryoid bodies (EBs). Although each of the previously introduced methods have been reported to induce approximately 20% HC-like cells and 10% HC-like cells in their respective populations in EB outgrowths at the end of the culture periods, the present combined method was able to generate approximately 30% HC-like cells expressing HC-related markers (myosin6, myosin7a, calretinin, α9AchR, Brn3c), which showed remarkable formation of stereocilia-like structures. Analysis of expressions of marker genes specific for cochlear (Lmod3, Emcn) and vestibular (Dnah5, Ptgds) cells indicated that our HIST2 method may lead to induction of cochlear- and vestibular-type cells. In addition, continuous Math1 induction by doxycycline without use of the HIST2 method preferentially induced cochlear markers with negligible effects on vestibular marker induction.

Barrington's nucleus: Neuroanatomic landscape of the mouse "pontine micturition center".

Barrington's nucleus (Bar) is thought to contain neurons that trigger voiding and thereby function as the "pontine micturition center." Lacking detailed information on this region in mice, we examined gene and protein markers to characterize Bar and the neurons surrounding it. Like rats and cats, mice have an ovoid core of medium-sized Bar neurons located medial to the locus coeruleus (LC). Bar neurons express a GFP reporter for Vglut2, develop from a Math1/Atoh1 lineage, and exhibit immunoreactivity for NeuN. Many neurons in and around this core cluster express a reporter for corticotrophin-releasing hormone (BarCRH ). Axons from BarCRH neurons project to the lumbosacral spinal cord and ramify extensively in two regions: the dorsal gray commissural and intermediolateral nuclei. BarCRH neurons have unexpectedly long dendrites, which may receive synaptic input from the cerebral cortex and other brain regions beyond the core afferents identified previously. Finally, at least five populations of neurons surround Bar: rostral-dorsomedial cholinergic neurons in the laterodorsal tegmental nucleus; lateral noradrenergic neurons in the LC; medial GABAergic neurons in the pontine central gray; ventromedial, small GABAergic neurons that express FoxP2; and dorsolateral glutamatergic neurons that express FoxP2 in the pLC and form a wedge dividing Bar from the dorsal LC. We discuss the implications of this new information for interpreting existing data and future experiments targeting BarCRH neurons and their synaptic afferents to study micturition and other pelvic functions.

Goblet cell carcinoids of the appendix: Tumor biology, mutations and management strategies.

Malignant neoplasms of the appendix are rare and represent less than 1% of gastrointestinal cancers. Goblet cell carcinoids (GCC) tumors are a distinctive group of heterogeneous appendiceal neoplasm that exhibit unique clinical and pathologic features. This review focuses on the current diagnostic procedures, pathogenesis, possible signaling mechanisms and treatment options for GCC. Perspectives for future research are discussed. The tumor likely arises from pluripotent intestinal epithelial crypt base stem cells. Previous findings of Notch signaling as a tumor suppressor in Neuroendocrine tumors may have a similar role in this tumor too. Loss of Notch signaling may be the driver mutation with other successive downstream mutations likely favors them into progressing and behavior similar to poorly differentiated adenocarcinoma with minimal neuroendocrine differentiation. A multidisciplinary approach is suggested for optimal outcomes. Surgery remains the main treatment modality. Simple appendectomy may be sufficient in early stages while right hemicolectomy is recommended for advanced tumors. Cytoreductive surgery with heated intraperitoneal chemotherapy may improve survival in a select few with metastatic peritoneal disease. These tumors have an unpredictable behavior even in early stages and local recurrence and delayed metastases may be seen. Lifelong surveillance is warranted.

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo.

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2'-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.

Development of intestinal organoids as tissue surrogates: cell composition and the epigenetic control of differentiation.

Intestinal organoids are multicellular crypt-like structures that can be derived from adult intestinal stem cells (ISCs), embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs). Here we show that intestinal organoids generated from mouse ESCs were enriched in ISCs and early progenitors. Treatment of these organoids with a γ-secretase inhibitor increased Math1 and decreased Hes1 expression, indicating Notch signaling regulates ISC differentiation in these organoids. Lgr5 and Tert positive ISCs constituted approximately 10% and 20% of the organoids. As found in native tissue, Lgr5 and Tert expressing cells resolved into two discreet populations, which were stable over time. Intestinal organoids derived from cancer-prone Apc(Min/+) mice showed similar numbers of ISCs, but had reduced Math1 expression, indicating a suppressed secretory cell differentiation potential (as found in intestinal tissue). Apc(Min/+) organoids were used to screen epigenetically active compounds for those that increased Math1 expression and organoid differentiation (including HDAC inhibitors, Sirtuin (SIRT) modulators and methyltransferase inhibitors). Broad-spectrum HDAC inhibitors increased both Math1 and Muc2 expression, indicating an ability to promote the suppressed secretory cell differentiation pathway. Other epigenetic compounds had a diverse impact on cell differentiation, with a strong negative correlation between those that activated the secretory marker Muc2 and those that activated the absorptive cell marker Fabp2. These data show that ESC-derived intestinal organoids can be derived in large numbers, contain distinct ISC types and can be used to screen for agents that promote cell differentiation through different lineage pathways.

Ectopic hair cell-like cell induction by Math1 mainly involves direct transdifferentiation in neonatal mammalian cochlea.

Math1, also known as Atoh1, is a basic helix-loop-helix transcription factor that plays a key role in hair cells (HCs) development. Previous studies have reported that Math1 gene transfer could induce the production of ectopic hair cell-like cells both in vitro and in vivo. Here, we focused on the mechanism of ectopic hair cell-like cellular differentiation from cells in the lateral epithelial ridge (LER) of cochlea with a human adenovirus serotype 5 (Ad5) vector encoding both Math1 and the reporter gene EGFP. Within the Ad5-EGFP-Math1 infection, hair-cell like cells could be detected in the LER. 5'-Bromo-2' deoxyuridine (BrdU) incorporation test results at different time points suggested that LER cells possessed high potential to proliferation, but they could not transdifferentiate into hair cells spontaneously. Almost all of Math1 induced hair cell-like cells were BrdU negative when BrdU incorporation occurred after Math1 expression. In conclusion, Math1 induced hair cell-like cells from LER cells mainly underwent direct trans-differentiation instead of mitosis of LER cells or newly hair cell-like cells.

The Construction of bFGF/Math1 Gene Vector and Its Expression in the Cochlea of Rat / 听力学及言语疾病杂志

Objective To construct the bFGF/Math1 f usion gene expression vector and to investigate its expression in the cochlea of rat. Methods Using the recombinant DNA technique to construct the bFGF/Math1 gene expression vector and the restriction enzyme analysis to ide ntify the correct construction. The mRNA expression was detected by the RT-PCR m ethod after being transfected into the cochlea of rat using lipofectin reagent. Results The recombinant was correct and the bFGF/Math1 wa s expressed in the cochlea of rat.Conclusion The PRK5-bFGF-Math1 eukaryotic expression plas mid was successfully constructed and the bFGF/Math1 was expressed in the mammali an cochlea.